Mutual and self-sensitivity among antibiotically active mutant derivatives from the inactive degenerate Aspergillus versicolor N5

Antibiotically active producer mutants derived from the spontaneous degenerate parent Aspergillus versicolor N₅ possessed not only mutual but also self-sensitive activity. The producer mutants, like the inactive parent, were only 3·5-fold less sensitive than the most sensitive unrelated organism, Trichophyton rubrum. The germination of spores is generally more sensitive than growth of vegetative cells. The antifungal spectrum of these mutual and self-sensitive mutants was fairly wide, unlike the host range specificity of bacteriocinogenic strains acting on organisms closely related to the producers. The self and mutual growth inhibitory principle was finally identified as the antibiotics mycoversilin and versilin in the case of producer mutants (N₅)₁₇ and N₅T₁₀(7), respectively, or V_x, an antibiotic of unknown molecular species, in the case of another producer mutant N₅T₁₀(8). Thus self-sensitivity, instead of self-resistance, of these antibiotically active mutant derivatives is a unique property among filamentous fungi in having simultaneously expressed two loci of contradictory functions, one for synthesis of, and the other for sensitivity towards, the same or related antibiotics.     

Mycelial growth and ochratoxin A production by Aspergillus section Nigri on simulated grape medium in modified atmospheres

Aims:  To evaluate the impact of modified atmosphere packaging on in vitro growth of Aspergillus carbonarius and Aspergillus niger , and possible effects on ochratoxin A (OTA) biosynthesis.
Methods and Results:  Ochratoxigenic isolates belonging to the species A. carbonarius and A. niger were grown on a synthetic grapejuice medium (SNM) and packaged in combinations of controlled O₂ (1% and 5%) and CO₂ levels (0% and 15%), and in air as a control. Colony diameters were recorded every 3 days up to 21 days, and OTA was analysed after 7, 14 and 21 days. The greatest reductions in mycelial growth rate were observed at 1% O₂ followed by 1% O₂/15% CO₂, whereas 5% O₂ stimulated the growth of all isolates. OTA production by A. carbonarius and A. niger isolates was minimized at 1% O₂/15% CO₂ and 1% O₂, respectively, after 7 days of incubation. Maximal OTA accumulation after 7 days was observed for all isolates in the control pack and at 5% O₂.
Conclusions:  Of the atmospheres tested, only 1% O₂ combined with 15% CO₂ consistently reduced fungal growth and OTA synthesis by A. carbonarius and A. niger .
Significance and Impact of the Study:  Storage under modified atmospheres is unlikely to be suitable as the sole method for OTA minimization and grape preservation; other inhibitory factors are necessary.     

Characterization of versilin-sensitive sites in self-sensitive producer and sensitive non-producer or unrelated organism

Studies on self-sensitivity of producer mutant vs. sensitivity of non-producer parent and unrelated organism showed that versilin inhibited spore germination and sporulation in the self-sensitive producer mutant, non-producer parent Aspergillus versicolor N₅ and the unrelated sensitive Trichophyton rubrum . Sporulation appeared to be more sensitive than spore germination. The inhibition of in vivo synthesis of protein was very marked, but inhibition of RNA and DNA was slight and moderate, respectively. Thus versilin was not specific in its action, but the principal sensitive site was protein synthesis, as further suggested by inhibition of polyU-directed in vitro synthesis of polyphenylalanine. The activation of leucine was unaffected, but the formation of leucyl-tRNA was severely inhibited in all three strains. The differences in sensitivities between the strains were the same, whether as whole cells or as cell-free extracts. Thus the nature of the sensitive site appeared to be identical in the self-sensitive producer and sensitive non-producer or unrelated organism.     

Wheat inoculation with Azospirillum brasilense Sp6 and some mutants altered in nitrogen fixation and indole-3-acetic acid production

Abstract Inoculation of wheat seedlings with Azospirillum brasilense Sp6 produced an increase in the number and length of the lateral roots as a plant response. Inoculation with a Nif⁻ mutant, A. brasilense SpF103, which is producer of indole-3-acetic acid (IAA), yielded a very similar plant response. However, inoculation with a Nif⁻ mutant, A. brasilense SpF57, which is a low producer of IAA, did not elitic any response from the plant. The data suggest that the root system response of wheat seedlings to bacterial inoculation is due mainly to production of auxin-type substances by the microorganism.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

工厂化生产海鲜菇菌包污染霉菌的鉴定及防治

对工厂化生产中海鲜菇菌包污染霉菌进行分离,根据霉菌的形态特征、培养性状及ITS序列分析,鉴定其为哈茨木霉、拟康氏木霉、脉孢霉、长枝木霉、黑曲霉、产红青霉和产黄青霉;在此基础上探讨了常用抑菌剂对霉菌的防治效果及对海鲜菇菌丝生长的影响。结果表明,质量浓度100 mg/L克霉灵对哈茨木霉、黑曲霉、产红青霉、产黄青霉、拟康氏木霉有强抑制作用,质量浓度100 mg/L多菌灵对长枝木霉、产红青霉、产黄青霉、拟康氏木霉有强抑制作用,二者对海鲜菇菌丝生长的抑制都比较弱。可为海鲜菇工厂化生产中污染霉菌的综合防治提供参考。     

The F1FO ATP synthase genes in Methanosarcina acetivorans are dispensable for growth and ATP synthesis

There is a long-standing discussion in the literature, based on biochemical and genomic data, whether some archaeal species may have two structurally and functionally distinct ATP synthases in one cell: the archaeal A₁A_O together with the bacterial F₁F_O ATP synthase. To address a potential role of the bacterial F₁F_O ATP synthase, we have exchanged the F₁F_O ATPase gene cluster in Methanosarcina acetivorans against a puromycin resistance cassette. Interestingly, the mutant was able to grow with no difference in growth kinetics to the wild type, and cellular ATP contents were identical in the wild type and the mutant. These data demonstrate that the F₁F_O ATP synthase is dispensable for the growth of M. acetivorans .     

Ecophysiological responses to simulated canopy gaps of two tree species of contrasting shade tolerance in elevated CO2

1. One-year-old seedlings of shade tolerant Acer rubrum and intolerant Betula papyrifera were grown in ambient and twice ambient (elevated) CO₂, and in full sun and 80% shade for 90 days. The shaded seedlings received 30-min sun patches twice during the course of the day. Gas exchange and tissue–water relations were measured at midday in the sun plants and following 20 min of exposure to full sun in the shade plants to determine the effect of elevated CO₂ on constraints to sun-patch utilization in these species.
2. Elevated CO₂ had the largest stimulation of photosynthesis in B. papyrifera sun plants and A. rubrum shade plants.
3. Higher photosynthesis per unit leaf area in sun plants than in shade plants of B. papyrifera was largely owing to differences in leaf morphology. Acer rubrum exhibited sun/shade differences in photosynthesis per unit leaf mass consistent with biochemical acclimation to shade.
4. Betula papyrifera exhibited CO₂ responses that would facilitate tolerance to leaf water deficits in large sun patches, including osmotic adjustment and higher transpiration and stomatal conductance at a given leaf-water potential, whereas A. rubrum exhibited large increases in photosynthetic nitrogen-use efficiency.
5. Results suggest that species of contrasting successional ranks respond differently to elevated CO₂, in ways that are consistent with the habitats in which they typically occur.     

Bactericidal and virucidal activity of the alkalophilic P395D/L241V/T343A mutant of vanadium chloroperoxidase

Aims:  Vanadium chloroperoxidase and its directed evolution mutant P395D/L241V/T343A were investigated for their antibacterial and antiviral potential at slightly alkaline pH and at a H₂O₂ concentration that is low compared to current nonenzymatic formulations.
Methods and Results:  Two bacteria (the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus ) and two viruses (the enveloped Herpes Simplex Virus and the nonenveloped Coxsackievirus B4) were incubated with the P395D/L241V/T343A mutant, 10 mmol l⁻¹ H₂O₂ and 100 mmol l⁻¹ Br⁻ at pH 8. Strong microbial reduction was observed and bactericidal and virucidal activities of the mutant were three to six orders of magnitude higher than for the wild-type enzyme.
Conclusions:  The P395D/L241V/T343A mutant of vanadium chloroperoxidase has a broad antimicrobial activity at alkaline conditions.
Significance and Impact of the Study:  For many disinfection formulations, antimicrobial activity at slightly alkaline pH values is required. To date, only the wild-type vanadium chloroperoxidase has been studied for its antibacterial activity, and only at acidic to neutral pH values. Its antiviral activity (e.g. useful for the cleaning of medical equipment) was not studied before. The observed activity for the alkalophilic P395D/L241V/T343A mutant is an important step forward in the application of this robust enzyme as a component in disinfection formulations.     

Enhancement of inulinase production by Aspergillus niger van Teighem

P. VISWANATHAN AND P.R. KULKARNI. 1995. Aspergillus niger van Teighem, a soil isolate, was mutagenized by u.v. light. A mutant strain with 3 times increased productivity was generated on exposure of the parent strain to u.v. for 15 min. This mutant on further exposure to u.v. yielded second generation mutants with only marginal increase in inulinase productivity with respect to the parent mutant, but the best yield of 377 U ml⁻¹ was considered promising for industrial use.     

Enhancement of β-galactosidase productivity of Aspergillus niger NCIM-616

A series of mutations was carried out with Aspergillus niger NCIM-616 as the parent strain. A mutant strain NG-4 with 28% increased β-galactosidase productivity was produced with N -methyl- N -nitro- N -nitrosoguanidine (NTG) at 1500µg ml^-1 concentration and exposure timeof 60 min. This mutant yielded a third generation u.v.-treated strain, UV—5 with a 117·6% increase in β-galactosidase productivity with respect to the parent strain.     

Uptake hydrogenase system in urd bean (Vigna mungo) Rhizobium in relation to nitrogen fixation

Twenty-five isolates of urd bean ( Vigna mungo ) Rhizobium were tested for the presence of an H₂-uptake system using triphenyl tetrazolium chloride reduction as the screening procedure. The isolates which reduced the dye rapidly at early stages of growth were found to recycle H₂ both in culture as well as in nodules. H₂-uptake positive, H₂-uptake negative strains and H₂-uptake negative mutants were compared on the basis of their effect on dry matter accumulation and N₂-fixation. Greenhouse experiments have shown the beneficial effect of the H₂-uptake positive system in nodules on N₂-fixation. Plants inoculated with H₂-uptake positive strains produced higher N content and dry matter over the plants inoculated with H₂-uptake negative strains.     

Tributyltin-resistant Methanothermobacter thermautotrophicus mutant with mutational substitutions in the A1A0-ATP synthase operon

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to tributyltin chloride (TBT) was isolated. TBT, the inhibitor of the A₀ domain of A₁A₀-ATP synthase, inhibits methanogenesis in the wild-type cells; however, the TBT-resistant mutant exhibited methanogenesis even in the presence of 800 μM TBT. ATP synthesis driven by methanogenic electron transport was markedly diminished in the mutant strain. While TBT profoundly inhibited ATP synthesis driven by methanogenic electron transport in the wild type, only a slight inhibition was observed in the mutant strain. These results suggested a modification in the ATP-synthesizing system of the mutant strain. The sequence of the complete A₁A₀-ATP synthase operon ( Mth952 – Mth961 ) in the wild-type and mutant strains was determined and compared. Three mutations leading to amino acid substitutions in two A₁A₀-ATP synthase subunits were identified – Val₃₃₈Ala in subunit A and Leu₂₅₂Ile and Ser₂₉₃Ala in subunit B. Moreover, this study revealed the differential expression of several proteins that may contribute to TBT resistance. The results imply that change of TBT sensitivities of TBT-resistant mutant is due to mutational substitutions in the A₁A₀-ATP synthase operon.     

Effect of gentian violet on the growth of the N and T RFLP types of the Aspergillus niger aggregate

Taxa included in the Aspergillus niger aggregate are difficult to distinguish by phenotypic characterization. In this work, the effect of gentian violet on the growth of the N and T RFLP types of A. niger aggregate strains has been investigated. In total, 105 strains from different sources and origins, including reference cultures and field isolates were studied. Type N and T RFLP patterns, ochratoxin A production and the effect of different concentrations of gentian violet on the growth were determined in these strains. Forty nine strains belonged to the N type and 56 strains to the T type. Sixteen out of the 105 strains assayed were OTA producers. All the OTA-producing species belonged to the RFLP type N and none of the T type strains was able to produce OTA. Approximately 90% of the N type strains grew in the presence of 25 ppm of gentian violet. Only five N type strains did not grow on this medium. One of these strains was A. niger ATCC 22343, a well documented induced mutant strain and the remaining four strains belonged to the new species A. brasiliensis. On the contrary, all the T type strains failed to grow on this medium after 3 days of incubation (sensitivity 89.79%; specificity 100%). The use of growth in gentian violet as an additional character for classification and identification purposes in this taxonomic group may be useful because no phenotypic methods have yet been found that can distinguish between these species.     

Characterisation of ovine Bordetella parapertussis isolates by analysis of specific endotoxin (lipopolysaccharide) epitopes, filamentous haemagglutinin production, cellular fatty acid composition and antibiotic sensitivity

Abstract Isolates of Bordetella parapertussis , recovered from sheep or man, were characterised by reaction with specific anti- Bordetella lipopolysaccharide monoclonal antibodies, production of filamentous haemagglutinin, fatty acid patterns, and antibiotic sensitivity. Generally, the isolates lay within one of four groups, with separation of the ovine isolates into two groups. Reactions with specific monoclonal antibodies against lipopolysaccharide separated the ovine isolates into these two groupings. Analysis of the cellular fatty acid compositions by cluster analysis differentiated between the human and the ovine strains and also showed variation within the ovine isolates. When the production of filamentous haemagglutinin was analysed in an ELIS A system, a similar pattern emerged. Varying concentrations of filamentous haemagglutinin (11–429 ng (mg total protein)⁻¹) were extracted from the human isolates and the one group of ovine isolates with no significant protein detected in the other ovine group. These studies demonstrate variation between and within B. parapertussis isolates recovered from two mammalian sources.     

Pichia angusta is an effective biocontrol yeast against postharvest decay of apple fruit caused by Botrytis cinerea and Monilia fructicola

The efficacy of eight isolates of Pichia angusta against three common postharvest pathogens of apple fruit was evaluated for the first time. All tested strains showed significant biocontrol activity against both Botrytis cinerea and Monilia fructicola , whereas efficacy against Penicillium expansum was poor. A leucine-auxotrophic mutant had no significant biocontrol activity against brown rot of apple, while the addition of 0.6–1.2 g L⁻¹ leucine in the fruit wound fully restored the biocontrol activity of this mutant against M. fructicola . Given the extremely well-developed classical and molecular genetics, the availability of genomic libraries, and its complete genomic sequence, this species can serve to elucidate the mechanisms related to biocontrol capacity.     

Acid phosphatase production by Aspergillus niger N402A in continuous flow culture

The production of acid phosphatases (E.C.3.1.3.2, ACPs) by Aspergillus niger N402A is regulated by specific growth rate, as well as phosphate availability and pH, as demonstrated by studies in continuous flow culture. Specific ACP activity was highest when A. niger was grown at pH 6.3 (64±8 U g⁻¹) or pH 2.8 (99±11 U g⁻¹), at a dilution rate of 0.07 h⁻¹ and phosphate concentrations below 0.46 mM. ACP production was growth correlated for specific growth rates between 0.07 and 0.13 h⁻¹. Four different ACPs, including two phytases, were produced by A. niger N402A. The ACP and the phytase with maximal activities at pH 5.5 were differentially expressed at different culture pH values, with greater production at low pH.     

Fusarium response to oxidative stress by H2O2 is trichothecene chemotype-dependent

The present study aims at clarifying the impact of oxidative stress on type B trichothecene production. The responses to hydrogen peroxide (H₂O₂) of an array of Fusarium graminearum and Fusarium culmorum strains were compared, both species carrying either the chemotype deoxynivalenol (DON) or nivalenol (NIV). In both cases, levels of in vitro toxin production are greatly influenced by the oxidative parameters of the medium. A 0.5 mM H₂O₂ stress induces a two- to 50-fold enhancement of DON and acetyldeoxynivalenol production, whereas the same treatment results in a 2.4- to sevenfold decrease in NIV and fusarenone X accumulation. Different effects of oxidative stress on toxin production are the result of a variation in Fusarium 's antioxidant defence responses according to the chemotype of the isolate. Compared with DON strains, NIV isolates have a higher H₂O₂-destroying capacity, which partially results from a significant enhancement of catalase activity induced by peroxide stress. A 0.5 mM H₂O₂ treatment leads to a 1.3- to 1.7-fold increase in the catalase activity of NIV isolates. Our data, which show the higher adaptation to oxidative stress developed by NIV isolates, are consistent with the higher virulence of these Fusarium strains on maize compared with DON isolates.